Ww.kaunoklinikos.lt

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2002, p. 3940–3946 0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.12.3940–3946.2002 Copyright 2002, American Society for Microbiology. All Rights Reserved.
Emergence of Tetracycline Resistance in Helicobacter pylori: Multiple Mutational Changes in 16S Ribosomal DNA and Other Genetic Loci Daiva Dailidiene,1 M. Teresita Bertoli,1,2 Jolanta Miciuleviciene,1,3 Asish K. Mukhopadhyay,1 Giedrius Dailide,1 Mario Alberto Pascasio,4 Limas Kupcinskas,3 and Douglas E. Berg1* Departments of Molecular Microbiology and Genetics, Washington University Medical School, St. Louis, Missouri 631101; Departamento de Ciencias Fisiolo´gicas, Escuela de Medicina, Universidad Dr. Jose´ Matias Delgado, La Libertad,2 and Servicio de Gastroenterología, Hospital Rosales, San Salvador,4 El Salvador; and Department of Gastroenterology, Kaunas University of Medicine, Kaunas, 3000, Lithuania3 Received 20 May 2002/Returned for modification 20 July 2002/Accepted 25 August 2002 Tetracycline is useful in combination therapies against the gastric pathogen Helicobacter pylori. We found 6
tetracycline-resistant (Tetr) strains among 159 clinical isolates (from El Salvador, Lithuania, and India) and
obtained the following four results: (i) 5 of 6 Tetr isolates contained one or two nucleotide substitutions in one
part of the primary tetracycline binding site in 16S rRNA (AGA965-967 [Escherichia coli coordinates] changed
to gGA, AGc, guA, or gGc [lowercase letters are used to represent the base changes]), whereas the sixth (isolate
Ind75) retained AGA965-967; (ii) PCR products containing mutant 16S ribosomal DNA (rDNA) alleles trans-
formed recipient strains to Tetr phenotypes, but transformants containing alleles with single substitutions
(gGA and AGc) were less resistant than their Tetr parents; (iii) each of 10 Tetr mutants of reference strain
26695 (in which mutations were induced with metronidazole, a mutagenic anti-H. pylori agent) contained the
normal AGA965-967 sequence; and (iv) transformant derivatives of Ind75 and of one of the Tetr 26695 mutants
that had acquired mutant rDNA alleles were resistant to tetracycline at levels higher than those to which either
parent strain was resistant. Thus, tetracycline resistance in H. pylori results from an accumulation of changes
that may affect tetracycline-ribosome affinity and/or other functions (perhaps porins or efflux pumps). We
suggest that the rarity of tetracycline resistance among clinical isolates reflects this need for multiple muta-
tions and perhaps also the deleterious effects of such mutations on fitness. Formally equivalent mutations with
small but additive effects are postulated to contribute importantly to traits such as host specificity and
virulence and to H. pylori’s great genetic diversity.
Helicobacter pylori is a genetically diverse gram-negative bac- binds tightly to a pocket in 16S rRNA, where it interferes terial species that chronically infects more than half of all sterically with binding of aminoacyl-tRNA to the ribosome A people worldwide and that can provoke development of peptic site and thereby blocks protein synthesis and bacterial growth.
ulcer disease and contribute to the risk of gastric cancer (for The tetracycline binding pocket has been defined with atomic reviews, see references 10 and 47). Several combination ther- resolution in the Thermus thermophilus ribosome and consists apies have been developed to eradicate this pathogen and cure of two domains in 16S rRNA: helix 34 and the loop next to or prevent associated diseases. First-line therapies usually in- helix 31 (6, 32). Resistance to tetracycline is common in many volve a proton pump inhibitor plus clarithromycin and either bacterial species. In Propionibacterium acnes this resistance is metronidazole or amoxicillin. They tend to fail in more than ascribed to a mutation affecting one specific position in the 10% of clinical trials, however, often because of bacterial re- tetracycline binding site (position G1058 in rRNA helix 34) in sistance to metronidazole, bacterial resistance to clarithromy- a remarkable 85% of tetracycline-resistant (Tetr) clinical iso- cin, and/or patient noncompliance. This led to the recommen- lates (34, 35). In most other species, however, resistance tends dation that a need for additional treatment be anticipated to be conferred by auxiliary genes carried on plasmids, trans- whenever H. pylori eradication is attempted and that tetracy- posons, or other mobile DNA elements and typically involves cline generally be used in such second-line (rescue) therapies proteins that variously (i) promote tetracycline efflux, (ii) bind (15, 27). In practice in developing countries, however, first-line to and change the 30S ribosomal subunit conformation and therapies often also involve tetracycline because metronida- thereby interfere with tetracycline-ribosome binding, or (iii) zole-resistant strains tend to be common and clarithromycin is cause enzymatic inactivation of the drug (9). Cases of resis- too expensive for most people (29; R. H. Gilman and A.
tance due to changes in chromosomally encoded porins or efflux pumps are also known (23, 25, 30).
Tetracycline is bacteriostatic, not bactericidal, against most Tetracycline resistance is uncommon among H. pylori clini- susceptible bacterial pathogens, H. pylori included (45). It cal isolates, at least in the United States and Europe. For example, no Tetr strain was found among more than 6,000 clinical isolates from the United States tested in D. Y. Gra- * Corresponding author. Mailing address: Department of Molecular ham’s laboratory (cited elsewhere [31] as unpublished data) or Microbiology, Campus Box 8230, Washington University Medical among any of nearly 1,900 strains from Portugal (8), The Neth- School, 4940 Parkview Place, St. Louis, MO 63110. Phone: (314) 362- 2772. Fax: (314) 362-1232 or (314) 362-7325. E-mail: BERG erlands (12), Sweden (19), Germany (40), or Australia (16).
Just 6 Tetr strains were found among 607 isolates tested from TETRACYCLINE RESISTANCE IN H. PYLORI Italy (38), Saudi Arabia (2), Bulgaria (5), and Spain (11).
these effects are additive: high-level, probably clinically signif- However, some 5 to 7% of strains from Japan and Korea (24) icant, resistance can emerge from combinations of multiple and a remarkable 59% of strains from Shanghai, People’s mutations with small but cumulative individual effects.
Republic of China (48), were reported to be Tetr. The last two reports, if confirmed, would indicate that tetracycline resis- tance is far more common in East Asia than in the West and MATERIALS AND METHODS
would be in accord with indications that East Asian and West- Bacterial strains and culture methods. The H. pylori reference strains used in
ern H. pylori gene pools differ markedly from one another (22, the present study were HPK5, which is highly transformable (39), and 26695, 49). One case of very high level tetracycline resistance in H. whose genome has been sequenced entirely (41). The 81 Lithuanian, 38 Salva-dorean, and 40 Indian clinical isolates screened for tetracycline resistance were pylori has been examined closely: Trieber and Taylor (42) re- from adults with gastric complaints who warranted endoscopy and were from cently found that resistance in a pair of closely related clinical patients of two of the authors (L.K. and M.A.P.) and Abhijit Chowdhury (29), isolates from the same patient (28) (in effect, one strain) was respectively. Genomic DNA from highly resistant Australian H. pylori strain due to a multisite mutation, a replacement of AGA by TTC in Aus108 (28), in which resistance is due to a change from AGA to TTC at 16SrDNA positions 965 to 967 (42), was kindly provided by C. Trieber and D.
the loop next to helix 31, one side of the tetracycline binding Taylor. All patients whose H. pylori strains were cultured had provided written pocket (16S rRNA positions 965 to 967) (42).
informed consent under protocols approved by local institutional human studies In the experiments described here we studied 6 H. pylori committees (Kaunas University of Medicine, Kaunas, Lithuania; Hospital Ro- strains that are resistant to low levels of tetracycline and that sales, San Salvador, El Salvador; and the Institute of Post Graduate Medical were found among 159 independent clinical isolates from El Education and Research, Calcutta, India).
H. pylori isolates were cultured on brain heart infusion agar (Difco) supple- Salvador, India, and Lithuania. The initial search for resistance mented with 7% horse blood, 0.4% IsoVitaleX, and the antibiotics amphotericin in this strain collection was motivated by a consideration that B (8 ␮g/ml), trimethoprim (5 ␮g/ml), and vancomycin (6 ␮g/ml) (referred to here tetracycline is available without prescription in each of these as BHI agar) and were incubated at 37°C under microaerobic conditions (10% three countries; that it is used extensively against cholera and CO2, 5% O2) as described previously (20). Metronidazole susceptibility and resistance were estimated by spotting aliquots of appropriately diluted bacterial other diarrheal diseases in India and El Salvador; and that it cultures on BHI agar with 8 ␮g of metronidazole per ml and scoring the efficiency had also been much used in Lithuania, often to avoid compli- of colony formation on metronidazole-containing agar relative to that on met- cations from other infections (even common colds, although ronidazole-free agar, as described previously (20).
such tetracycline use decreased as other antimicrobial agents To score tetracycline susceptibility or resistance, cells growing exponentially became more available after independence in 1990). These six on tetracycline-free BHI agar were suspended in phosphate-buffered salinebuffer, and about 106 cells were spotted on medium with 2 ␮g of tetracycline per Tetr strains were studied to understand the mechanisms that ml (tet2 medium), and on tetracycline-free medium as a control. An isolate was underlie the resistance phenotype and why tetracycline resis- considered resistant if any bacterial growth was observed after 1 week of incu- tance is so uncommon, at least in Western H. pylori popu- bation. The level of resistance of Tetr isolates was also examined more closely by lations. Three findings contributed importantly to our study an efficiency of colony formation (efficiency of plating) test, in which a series of10-fold dilutions of cell suspensions was prepared; and 10 ␮l of each dilution was design. First, resistance of H. pylori to the macrolide clarithro- spotted on freshly prepared BHI agar containing various concentrations of tet- mycin results from any of several specific single nucleotide racycline (e.g., 0, 0.5, 1.0, 2.0, and 4 ␮g/ml). When accurate estimates of very low substitutions in 23S rRNA; mutant ribosomal DNA (rDNA) frequencies of colony formation (Ͻ10Ϫ6) were needed, culture aliquots were alleles are easily moved to new strains by transformation and spread directly on the surface of an entire plate of tetracycline-containing BHI selection for resistance. Most such clarithromycin-resistant agar rather than spotted in a small area.
Levels of resistance were more difficult to quantify with tetracycline than with (Clar) transformants contain only the mutant allele (which metronidazole. For example, small increases in near-threshold tetracycline con- encodes resistance), even though H. pylori contains two copies centrations tended to cause progressive decreases in the fraction of cells able to of each rRNA gene (43, 44, 46). Thus, a recessive rDNA allele form colonies and also in colony size (culminating in invisibility without the use can be acquired, rendered homozygous, and expressed rela- of a magnifying glass, independent of the time of incubation). This contrastedwith the abrupt changes in colony-forming efficiency, but little change in colony tively efficiently in H. pylori. Second, resistance to metronida- size, with changes in concentration that were seen in similar analyses of metro- zole, which is common in many H. pylori populations, usually nidazole susceptibility (20). As a consequence, estimates of a strain’s colony- results from inactivation of chromosomal genes for nitroreduc- forming efficiency at a given tetracycline concentration (in effect, resistance level) tases (rdxA and, in some cases, frxA) that mediate conversion varied slightly from trial to trial. However, the same rank order of resistance of metronidazole from a prodrug to a bactericidal agent (20, levels for different clinical isolates or transformants was observed in repeatedparallel tests. Variations in test results may reflect the fact that tetracycline is 21). Thus, unlike drug resistance in many pathogens, resistance more bacteriostatic than bactericidal and the fact that it is somewhat unstable in H. pylori usually involves only mutant alleles of normal during incubation; any effects of subtle differences in bacterial physiologic states chromosomal genes, not mobile DNA element-borne auxiliary on the effectiveness of tetracycline inhibition would also contribute to variability.
resistance determinants. Third, tetracycline resistance in P. Given these observations, for parsimony in assembling the results, a strain wasconsidered to still be rather resistant to any level of tetracycline that allowed acnes is often due to a mutation in the tetracycline binding colony formation with at least 10% the efficiency of that seen with the next lower pocket in 16S rRNA (34, 35), as noted above.
tetracycline level; and it was considered susceptible to the next higher tetracy- The six Tetr clinical isolates studied here were each less cline concentration used if that concentration caused at least a 1,000-fold de- resistant than the Tetr strain that Trieber and Taylor (42) crease in the efficiency of colony formation. It is noteworthy that for several characterized. Five of the six contained one or two nucleotide isolates tested, cells from minute colonies recovered from media with partiallyinhibitory concentrations of tetracycline had susceptibility profiles that matched changes in the loop next to helix 31, which forms part of the those of the parental cell line, which had not been grown with tetracycline. These tetracycline binding pocket, whereas the sixth did not. Further cells seemed not to be spontaneous resistant mutants, which is quite different tests showed that tetracycline resistance in H. pylori can be from the pattern seen with cells recovered from media with partially inhibitory complex and multifactorial and can involve various mutant concentrations of metronidazole (20, 21).
PCR and DNA sequencing. DNAs were prepared from confluent BHI agar
alleles of 16S rRNA genes or non-rDNA determinants, or plate cultures derived from single colonies or from pools of colonies, as appro- both. Most individual mutations have only weak effects, but priate, with QIAamp Tissue DNA extraction kits (Qiagen Corporation, Chats- worth, Calif.). To detect changes in 16S rRNA sequences associated with tetra-cycline resistance, nearly full-length (1.4-kb) segments of 16S rRNA genes wereamplified by PCR with primers 16S-F (5Ј-CGGTTACCTTGTTACGACTTCAC) and 16S-R (5Ј-TATGGAGAGTTTGATCCTGGCTC). The PCR was car-ried out in 50-␮l volumes containing 10 ng of genomic DNA, 10 pmol of eachprimer, 1 U of Biolase (Taq DNA polymerase equivalent) from Midwest Scien-tific (St. Louis, Mo.), and 0.25 mmol of each deoxynucleoside triphosphate instandard PCR buffer. The reaction mixtures were preincubated for 2 min at 94°Cand were then subjected to 30 cycles of 94°C for 40 s, 52°C for 40 s, and 72°C for2 min, with a final elongation step of 72°C for 10 min. PCR fragments werepurified for sequencing with a QIAquik PCR purification kit (Qiagen Corpora-tion). PCR-amplified 16S rDNAs were sequenced by using primers 16S-R and16S-F and also primer 16S-F2 (5Ј-TCAAGCCTAGGTAAGGTTCTTCG). Se-quencing reactions were carried out with a Big Dye Terminator cycle sequencingkit (PE Applied Biosystems, Foster City, Calif.), and products were run onApplied Biosystems automated sequencers in the Washington University Mo-lecular Microbiology core facility.
Nucleotide sequence accession numbers. The 16S rDNA sequences deter-
mined in the present study have been deposited in GenBank under accessionnumbers AF535194 to AF535200 (for strains Sal05, SalI0, Lit69, Lit76, Lit81,HPK5, and Ind75, respectively).
Tetr clinical isolates and mutant 16S rRNA genes. Prelimi-
nary tests showed that individual cells from diluted cultures of several reference H. pylori strains, including strains 26695 and HPK5 (39, 41), did not form colonies on freshly prepared medium with 2 ␮g of tetracycline per ml (tet2 medium) under FIG. 1. Diagram of polymorphic sites in 16S rDNAs of Tets refer- our conditions but did form colonies on media with lower ence strains of H. pylori, Tetr clinical isolates, and Tetr transformant tetracycline concentrations (0.5 or 1 ␮g/ml) about as efficiently derivatives of HPK5 (Tets) and Ind75 (low-level Tetr) generated as detailed in the text and in GenBank accession numbers AF535194 to as they did on tetracycline-free medium. Cells from colonies of AF535200. The positions in the rRNA sequence (adapted to E. coli several strains that had grown on tet0.5 or tet1 medium re- 16S rRNA coordinates; GenBank accession number AE000474) are mained unable to grow on tet2 medium, which indicates that listed vertically. For example, the 1st column of sequence corresponds they were not mutants. A set of 38 clinical isolates from El to position 92; the 5th column corresponds to position 360; and the 9th, 10th, and 11th columns (boldfaced) correspond to positions 965, 966, Salvador, 81 isolates from Lithuania, and 40 isolates from and 967, the sites of mutations inferred to contribute to tetracycline India (159 isolates in total) was then screened for growth on resistance (lowercased), as discussed in the text. Periods indicate base tet2 medium. Five strains (two Salvadorean and three Lithua- substitutions relative to the 16S rDNA sequence. Dashes indicate nian strains) were judged to be resistant. They formed colonies absent nucleotides (1-bp deletions). Empty spaces indicate sequences with nearly 100% efficiency on tet2 medium, whereas most not determined. clin, clinical isolate. HPK5 and Ind75 used as recipi- ents for transformation using rDNAs from Tetr clinical isolates are other clinical isolates screened could not grow on this medium italicized. Highly resistant strain Aus108/9, examined by others (42), is (no colonies per 106 cells plated). One Indian strain (strain included for comparison. Not shown here is position 1024, which is Ind75) formed minute colonies efficiently on tet1.5 medium, 1024T in HPK5 but 1024C in most H. pylori strains, including all Tetr unlike any of the other 39 Indian strains screened, and thus was clinical isolates identified in our studies.
also designated resistant. For each of these six strains, how- ever, the speed of colony development was reduced on tet1.5 segment that cooperates with helix 34 to form the primary or tet2 medium (colonies could be seen without magnification tetracycline binding site. Two strains were doubly mutant at ϳ5 days on tet2 medium, whereas they could be seen with- (AGA965-967 changed to gtA and gGc [lowercase letters are out magnification at ϳ3 days on tetracycline-free medium).
used to represent the base changes]); three others contained The three Lithuanian and two Salvadorean Tetr clinical iso- just single nucleotide substitutions at this same site (AGA lates also made minute colonies on tet4 medium, but generally changed to gGA or AGc); and the sixth (strain Ind75) con- with efficiencies of only 1% or less, and they became visible tained the canonical AGA sequence at this site. In contrast, only after 6 days of incubation. Three of these six Tetr strains each of 33 H. pylori 16S rRNA entries found in a database (strains Lit69, Lit76, and Lit81) were susceptible to metroni- search in June 2002 (excluding three entries for the Tetr strains dazole, which contrasts with the reported metronidazole resis- [with ttc in place of AGA] of Trieber and Taylor [42]) con- tance of all Tetr strains in two East Asian H. pylori collections tained AGA at this site. Several other differences in the rDNA sequences of Tetr isolates and tetracycline-susceptible (Tets) Given the many Tetr P. acnes isolates in which resistance was reference strains were also found, but none was consistently due to substitution at 16S rRNA position G1058 (helix 34), found among all Tetr isolates (Fig. 1). It is striking, however, which forms part of the tetracycline binding pocket (34, 35), we that none of the Tetr H. pylori clinical isolates contained se- amplified by PCR and sequenced 16S rRNA genes from each quence changes at or near G1058, the residue that is so im- of these six new Tetr H. pylori strains. The sequences of five of portant in tetracycline resistance in P. acnes, nor were changes them differed from that of the canonical wild-type strain in the found in other, lower-affinity tetracycline binding sites (32).
loop adjacent to helix 31 (positions 965 to 967) (Fig. 1), a 16S rRNA sequence changes underlie much but not all of
TETRACYCLINE RESISTANCE IN H. PYLORI the tetracycline resistance in clinical isolates. To further test if
TABLE 1. Level of tetracycline resistancea is determined by a nucleotide substitutions in 16S rRNA contributed to resis- combination of background genotype and 16S rDNA allele tance, PCR-amplified 16S rDNAs from each of the six Tetr isolates were used to transform Tets strain HPK5, and resistant transformants were selected on tet2 medium. Single discrete colonies were obtained at frequencies of about 10Ϫ4 in 4 days after transformation with DNAs containing double substitu- tions from positions 965 to 967 (strains Lit76 and Lit81); much smaller, barely visible colonies were obtained only at 6 days after transformation with DNAs from the three strains with a Tetracycline resistance levels were difficult to define with precision, because single substitutions (strains Lit69, Sal05, and SalI0); and no increases in tetracycline levels resulted in progressive decreases in colony size colonies were obtained by using the PCR products from the and efficiency of colony formation, as detailed in Materials and Methods.
16S rDNA from strain Ind75 (which contains AGA at positions The tetracycline concentration that still allowed pretty good growth. The levels tested by efficiency-of-plating assays were 0 0.5, 1.5 or 2, 4, 6, 8, 12, 16, 32, 965 to 967) or any of three Tets control strains. The expected 64 and 128 ␮g of tetracycline per ml. The resistance levels given here are the rDNA alleles with changes at positions 965 to 967 were found highest levels that cause no more than a 10-fold decrease in efficiency of colony formation relative to that caused by the next highest level of tetracycline, as by sequencing in each of the 10 Tetr transformants tested (two detailed in Materials and Methods. WT, wild type.
from each of the five donor DNAs; Fig. 1). Nearby sequences that distinguished the Tetr clinical isolates from the HPK5 recipient were also present in many transformants; these may ments. We therefore mutagenized cells of strains 26695 by reflect normal genetic linkage, not mechanistic involvement in growing them on medium containing low concentrations (2 or 3 ␮g per ml) of metronidazole, a condition in which many cells Although H. pylori contains two copies of the 16S rRNA are killed by this anti-H. pylori drug and in which survivors gene, inspection of sequence tracings showed that 9 of the 10 contain a markedly increased frequency of mutations (36, 37).
transformants were homozygous (contained only the mutant Some 10Ϫ6 to 10Ϫ5 of cells harvested from metronidazole- allele), indicating that the emergence of transformants with a containing medium formed colonies on tet2 medium, whereas resistant phenotype typically involves acquisition of the mutant Ͻ10Ϫ8 cells harvested from metronidazole-free medium sequence by both 16S rRNA gene copies, presumably one after formed colonies. These estimates of colony-forming efficiency the other. One transformant generated with Lit081 DNA was were imprecise, however, because colony sizes ranged from heterozygous, which, although not understood (susceptibility barely perceptible to near normal (diameter, Ն1 mm) even was expected to be dominant), is reminiscent of the finding of after 7 days of incubation. Six to 7 days of incubation was also occasional heterozygous transformants generated with 23S needed for these Tetr colonies to appear on the initial tet2 rDNA alleles that confer clarithromycin resistance (18, 44). In selection plates, which is about 2 days longer than the time summary, these results show that changes in the 16S rRNA needed for the emergence of Tetr transformants. However, no sequence can confer resistance to tetracycline in H. pylori. The colonies or background growth was detected after parallel plat- weaker Tetr phenotype of transformants containing rDNA al- ing of 108 cells from a nonmutagenized control culture of the leles with single substitutions relative to the Tetr phenotype of same strain on tet2 medium. Further tests with 16 Tetr mutants their parental clinical isolates and the inability of rDNA se- selected on tet2 plates indicated that their resistant phenotype quences from Tetr isolate Ind75 to confer resistance on the was retained after subculture on tetracycline-free medium.
Tets recipient strain both imply that non-rRNA determinants Two of eight and five of eight mutants from cultures pregrown also contribute to tetracycline resistance in H. pylori.
with 2 and 3 ␮g of metronidazole per ml, respectively, formed In follow-up experiments, weakly Tetr clinical isolate Ind75 colonies on tet6 medium, albeit less rapidly and some 100- to (grown on tet1.5 medium) was transformed with PCR-ampli- 1,000-fold less efficiently than on tet2 medium; the other nine fied 16S rDNA from Tetr strains Lit76 and Aus108, and colo- mutants grew well on tet2, only weakly on tet4 medium, and nies that grew well on tet3 medium were selected. Several not at all on tet6 medium. Thus, intrinsic levels of tetracycline representative transformants made with Lit76 DNA (AGA to resistance varied among the new Tetr mutants.
gtA) grew, albeit weakly, on tet8 but not on tet16 medium; a The sequence of the 0.7-kb segment near the 3Ј end of 16S transformant made with a PCR product from Aus108 (AGA to rRNA (containing the helix 31 loop and the helix 34 compo- ttc) grew on tet32 but not on tet64 in the same experiment nents of the primary tetracycline binding pocket) was deter- (Table 1). The 16S rDNAs of representative transformants mined as described above for 10 of the metronidazole-induced were sequenced and found to contain the expected gtA965-967 Tetr mutants (5 that grew on tet6 medium and 5 that were less and ttc965-967 alleles. Because these transformants were more resistant, that is, that grew on tet2 medium and to some extent resistant than either parent, we infer that the naturally occur- on tet4 medium). None of these Tetr mutants contained ring 16S rDNA of Lit76 or Aus108 and the non-rRNA deter- changes in or near the primary tetracycline binding site or in minants of Ind75 contribute additively to resistance pheno- any of the potential secondary binding sites (6, 32) in this 3Ј half of the 16S rDNA sequence. The sequences of much of the Tetr mutants generated in culture. To further assess poten-
other (5Ј) half of the 16S rRNA gene (which contains one arm tial contributions of non-rDNA versus rDNA gene mutations of one of the secondary tetracycline binding sites [32]) were to tetracycline resistance, we selected and characterized Tetr also determined from two of the most resistant mutants. Again, mutant derivatives of reference strain 26695. No (Ͻ10Ϫ8) no sequence changes likely to affect tetracycline binding were spontaneous Tetr mutants were found in preliminary experi- found (GenBank accession numbers AF535194 to AF535198 and AF535200). This implies that each of these 10 laboratory- That other (non-rRNA) genes can also contribute to tetra- generated Tetr mutants had arisen as a result of changes in cycline resistance was indicated by findings that (i) the level of resistance conferred by rDNA alleles with single substitutions One mutant that grew on tet4 medium, albeit very weakly, in the critical trinucleotide from positions 965 to 967 after was transformed with PCR products containing the Lit76 transformation of strain 26695 was weaker than the resistance (AGA to gtA) and Aus108 (AGA to ttc) alleles, and transfor- of parental Tetr clinical isolates, (ii) one weakly resistant clin- mants that grew well on tet4 medium were selected and puri- ical isolate had no mutation in its 16S rRNA tetracycline bind- fied by restreaking them on fresh tet4 medium before analyses ing site, and (iii) the rDNA sequences of laboratory-generated of their rDNA alleles. The gtA and ttc alleles each inactivate a Tetr mutant derivatives of strain 26695 were also unchanged BstBI site (positions 959 to 965; TTCGAA to TTCGAg or from those of their Tets parent. In addition, introduction of TTCGAt due to the changes to AGA at positions 965 to 967).
mutant rDNA alleles into either the clinical isolate or a 26695 Restriction analysis of PCR-amplified 16S rDNAs from several mutant with non-rRNA-based resistance resulted in higher- representative transformants showed that the mutant alleles level tetracycline resistance than the level of resistance of ei- had indeed been acquired and that the transformants were ther parent. The two types of resistance determinants (non- homozygous in each case (data not shown). Representative rRNA- and rRNA binding site-based resistance) are inferred transformants generated with Lit76 DNA grew weakly on tet32 to contribute to resistance by complementary mechanisms.
medium but not on tet64 medium, whereas those generated Possible contributors to non-rRNA-based resistance include with Aus108 DNA were slightly more resistant in that they (i) various porin genes (1, 4, 30, 50); (ii) the putative tetA efflux grew on tet64 medium but not on tet128 medium (Table 1).
gene, HP1165 (42), homologs of which are present in several The corresponding transformants of the ancestral 26695 wild- cryptic H. pylori plasmids (13, 33); and (iii) several other pu- type strain grew only on tet12 medium but not on tet16 me- tative efflux genes (arcB family HP0607, HP0969, and dium (strain Lit076) and on tet32 medium but not on tet64 HP1329), which are unrelated to HP1165 (tetA) (42). Regard- medium (strain Aus108) (Table 1). Because these transfor- less of the exact nature of these determinants, their apparently mants were also more resistant than either parent, we infer low abundance in H. pylori clinical isolates would be explained once again that higher-level tetracycline resistance can be if they decrease fitness and thus tend to be contraselected achieved through the additive effects of resistance mutations in during human infection. More generally, we suggest that tet- 16S rDNA plus additional mutations at one or more unknown racycline resistance may be uncommon in H. pylori for three reasons: (i) the rarity of mobile DNA elements carrying aux- iliary resistance genes; (ii) a need for multiple very specific DISCUSSION
nucleotide sequence changes in rDNA and/or other genes to achieve high resistance; and (iii) the effects of resistance mu- We have studied the mechanisms of tetracycline resistance, tations, whether in rDNAs or elsewhere, on bacterial fitness.
a trait with a potentially high degree of clinical significance that Our results illustrate that accumulations of genetic determi- is exhibited by a small minority of H. pylori clinical isolates. In nants with small individual effects can, in the aggregate, signif- five of the six Tetr isolates studied, resistance could be ascribed icantly affect H. pylori phenotypes. They suggest that mutant H. at least in part to one or two substitutions in a three-nucleotide pylori strains with even slight tetracycline resistance are fa- loop region that forms part of tetracycline’s primary binding vored whenever inhibitory concentrations of tetracycline are site in the ribosome, positions 965 to 967 in 16S rRNA. Studies encountered. This may occur repeatedly during typical multi- of isogenic strains generated by the transformation of mutant year chronic infections, especially in the many countries in rDNA alleles into a uniform genetic background showed that which tetracycline use is not regulated by prescription. Because single nucleotide substitutions conferred only weak resistance; tetracycline is primarily bacteriostatic, resistance need not be that double substitutions conferred a somewhat higher level of very strong to be advantageous: any mutant whose growth is resistance; and that the triple substitution, which others (42) less inhibited by tetracycline than its parent’s should tend to had first analyzed, conferred high-level resistance, as expected.
outgrow the parent whenever this drug is present, quite inde- Thus, the strength of resistance seems proportionate to the pendent of the dose or the duration of treatment. Even slight severity of change (deformation) of tetracycline’s primary ri- inhibition of such mildly resistant mutants during subsequent bosome binding site. None of the Tetr clinical isolates had exposures to tetracycline will foster the stepwise emergence of mutations in the other (helix 34) arm of this binding site, nor derivatives with higher-level resistance. This could be due to a had specific mutagenesis by others (42) of the position in that series of additional mutations and/or to interstrain transfer of arm that underlies most resistance in P. acnes (position G1058) DNA with already formed resistance determinants. That resis- yielded Tetr H. pylori. The different outcomes in H. pylori and tance is nevertheless uncommon in current H. pylori popula- P. acnes may reflect subtle differences in ribosome structure.
tions suggests an additional mitigating force. In particular, as For example, given the importance of tetracycline’s primary noted above, many resistance determinants may be deleterious binding site for aminoacyl-tRNA binding to the ribosome (6, when no tetracycline is present, although such effects are often 32), perhaps no change in the helix 34 arm of H. pylori can overcome by yet additional compensatory mutations (3). Even- significantly diminish tetracycline binding yet still allow ribo- tually, this series of adaptive mutations may culminate in somes to act effectively in protein synthesis; or, given multiva- strains that are sufficiently resistant to render tetracycline- lent tetracycline binding, perhaps no single change in this arm based rescue therapy ineffective and that yet retain their viru- alters tetracycline binding sufficiently to confer detectable re- lence. This was probably the case with the triple mutant strain (strain Aus108 [AGA965ttc]) (42) that was isolated from an TETRACYCLINE RESISTANCE IN H. PYLORI elderly peptic ulcer patient after several failed tetracycline- Valle, M. Yang, H. P. Wirth, G. I. Perez-Perez, and M. J. Blaser. 1999. Host
based courses of anti-H. pylori therapy (28).
specificity of Helicobacter pylori strains and host responses in experimentally challenged nonhuman primates. Gastroenterology 116:90–96.
More generally, we suggest that quantitative genetic deter- 14. Fischbach, L. A., K. J. Goodman, M. Feldman, and C. Aragaki. 2002.
minants, formally equivalent to those studied here, contribute Sources of variation of Helicobacter pylori treatment success in adults world- importantly to bacterium-human host interactions and the wide: a meta-analysis. Int. J. Epidemiol. 31:128–139.
15. Gisbert, J. P., and J. M. Pajares. 2001. Helicobacter pylori therapy: first-line
great genetic diversity of H. pylori strains worldwide. We pro- options and rescue regimen. Dig. Dis. 19:134–143.
pose that the human gastric mucosa be considered a rugged 16. Grove, D. I., G. Koutsouridis, and A. G. Cummins. 1998. Comparison of
evolutionary landscape (7), diverse and challenging to each culture, histopathology and urease testing for the diagnosis of Helicobacter pylori gastritis and susceptibility to amoxycillin, clarithromycin, metronida- strain that infects it. Any differences among us in traits that zole and tetracycline. Pathology 30:183–187.
could be important to individual H. pylori strains (13) and any 17. Hofreuter, D., and R. Haas. 2002. Characterization of two cryptic Helico-
bacter pylori plasmids: a putative source for horizontal gene transfer and gene changes in gastric physiology caused by our host response to shuffling. J. Bacteriol. 184:2755–2766.
chronic infection (26) should each select for adjustments in 18. Hulten, K., A. Gibreel, O. Skold, and L. Engstrand. 1997. Macrolide resis-
bacterial phenotype. Many such adjustments will be quantita- tance in Helicobacter pylori: mechanism and stability in strains from clar- ithromycin-treated patients. Antimicrob. Agents Chemother. 41:2550–2553.
tive and, due to an accumulation of genetic changes with small 19. Jaup, B. H., A. Brandberg, B. Stenquist, and A. Norrby. 1998. Antibiotic
but additive individual effects, will result in an evolutionary resistance among strains of Helicobacter pylori in Gothenburg. Bacteria re- tinkering equivalent to that leading to the emergence of tetra- sistant to metronidazole. Lakartidningen 95:279–281.
20. Jeong, J. Y., A. K. Mukhopadhyay, J. K. Akada, D. Dailidiene, P. S. Hoffman,
and D. E. Berg. 2001. Roles of FrxA and RdxA nitroreductases of Helico-
bacter pylori in susceptibility and resistance to metronidazole. J. Bacteriol.
ACKNOWLEDGMENTS
183:5155–5162.
21. Jeong, J. Y., A. K. Mukhopadhyay, D. Dailidiene, Y. Wang, B. Velapatin˜o,
We thank Arvydas Janulaitis for encouragement of studies of the H. R. H. Gilman, A. J. Parkinson, G. B. Nair, B. C. Y. Wong, S. K. Lam, R.
pylori strains of Lithuania; Simanti Datta and Abhijit Chowdhury for Mistry, I. Segal, Y. Yuan, H. Gao, T. Alarcon, M. L. Brea, Y. Ito, D. Kersu-
lyte, H.-K. Lee, Y. Gong, A. Goodwin, P. S. Hoffman, and D. E. Berg. 2000.
participation in early experiments; and Abhijit Chowdhury, Robert H.
Sequential inactivation of rdxA (HP0954) and frxA (HP0642) nitroreductase Gilman, G. Balakrish Nair, Catherine Trieber, and Diane Taylor for genes cause moderate and high-level metronidazole resistance in Helicobac- discussion and communication of unpublished results or provision of ter pylori. J. Bacteriol. 182:5082–5090.
22. Kersulyte, D., A. K. Mukhopadhyay, B. Velapatino, W. Su, Z. Pan, C. Garcia,
This research was supported by grants from the U.S. Public Health V. Hernandez, Y. Valdez, R. S. Mistry, R. H. Gilman, Y. Yuan, H. Gao, T.
Service (grants AI38166, AI49161, DK53727, and P30 DK52574).
Alarcon, M. Lopez-Brea, G. B. Nair, A. Chowdhury, S. Datta, M. Shirai, T.
M.T.B. is the recipient of an ASM Fellowship for Latin America.
Nakazawa, R. Ally, I. Segal, B. C. Wong, S. K. Lam, F. O. Olfat, T. Boren, L.
G.D. is a Ph.D. candidate at Kaunas University of Medicine, Kau- Engstrand, O. Torres, R. Schneider, J. E. Thomas, S. Czinn, and D. E. Berg.
2000. Differences in genotypes of Helicobacter pylori from different human populations. J. Bacteriol. 182:3210–3218.
23. Koutsolioutsou, A., E. A. Martins, D. G. White, S. B. Levy, and B. Demple.
REFERENCES
2001. A soxRS-constitutive mutation contributing to antibiotic resistance in a 1. Alm, R. A., J. Bina, B. M. Andrews, P. Doig, R. E. Hancock, and T. J. Trust.
clinical isolate of Salmonella enterica (serovar Typhimurium). Antimicrob.
2000. Comparative genomics of Helicobacter pylori: analysis of the outer Agents Chemother. 45:38–43.
membrane protein families. Infect. Immun. 68:4155–4168.
24. Kwon, D. H., J. J. Kim, M. Lee, Y. Yamaoka, M. Kato, M. S. Osato, F. A.
2. Al-Qurashi, A. R., F. El-Morsy, and A. A. Al-Quorain. 2001. Evolution of
El-Zaatari, and D. Y. Graham. 2000. Isolation and characterization of tet-
metronidazole and tetracycline susceptibility pattern in Helicobacter pylori at racycline-resistant clinical isolates of Helicobacter pylori. Antimicrob. Agents a hospital in Saudi Arabia. Int. J. Antimicrob. Agents 17:233–236.
Chemother. 44:3203–3205.
3. Andersson, D. I., and B. R. Levin. 1999. The biological cost of antibiotic
25. Levy, S. B. 1992. Active efflux mechanisms for antimicrobial resistance.
resistance. Curr. Opin. Microbiol. 2:489–493.
Antimicrob. Agents Chemother. 36:695–703.
4. Bina, J. E., R. A. Alm, M. Uria-Nickelsen, S. R. Thomas, T. J. Trust, and
26. Mahdavi, J., B. Sonden, M. Hurtig, F. O. Olfat, L. Forsberg, N. Roche, J.
R. E. Hancock. 2000. Helicobacter pylori uptake and efflux: basis for intrinsic
Angstrom, T. Larsson, S. Teneberg, K. A. Karlsson, S. Altraja, T. Wadstrom,
susceptibility to antibiotics in vitro. Antimicrob. Agents Chemother. 44:248–
D. Kersulyte, D. E. Berg, A. Dubois, C. Petersson, K. E. Magnusson, T.
Norberg, F. Lindh, B. B. Lundskog, A. Arnqvist, L. Hammarstrom, and T.
5. Boyanova, L., I. Stancheva, Z. Spassova, N. Katzarov, I. Mitov, and R.
Boren. 2002. Helicobacter pylori SabA adhesin in persistent infection and
Koumanova. 2000. Primary and combined resistance to four antimicrobial
chronic inflammation. Science 297:573–578.
agents in Helicobacter pylori in Sofia, Bulgaria. J. Med. Microbiol. 49:415–418.
27. Malfertheiner, P., F. Megraud, C. O’Morain, A. P. Hungin, R. Jones, A.
6. Brodersen, D. E., W. M. Clemons, Jr., A. P. Carter, R. J. Morgan-Warren,
Axon, D. Y. Graham, and G. Tytgat. 2002. Current concepts in the manage-
B. T. Wimberly, and V. Ramakrishnan. 2000. The structural basis for the
ment of Helicobacter pylori infection—the Maastricht 2–2000 Consensus action of the antibiotics tetracycline, pactamycin, and hygromycin B on the Report. Aliment. Pharmacol. Ther. 16:167–180.
30S ribosomal subunit. Cell 103:1143–1154.
28. Midolo, P. D., J. R. Lambert, T. G. Kerr, and W. Tee. 1999. In vitro synergy
7. Burch, C. L., and L. Chao. 1999. Evolution by small steps and rugged
between ranitidine bismuth citrate and tetracycline or clarithromycin against landscapes in the RNA virus phi6. Genetics 151:921–927.
resistant strains of Helicobacter pylori. Eur. J. Clin. Microbiol. Infect. Dis.
8. Cabrita, J., M. Oleastro, R. Matos, A. Manhente, J. Cabral, R. Barros, A. I.
18:832–834.
Lopes, P. Ramalho, B. C. Neves, and A. S. Guerreiro. 2000. Features and
29. Mukhopadhyay, A. K., D. Kersulyte, J. Y. Jeong, S. Datta, Y. Ito, A.
trends in Helicobacter pylori antibiotic resistance in Lisbon area, Portugal Chowdhury, S. Chowdhury, A. Santra, S. K. Bhattacharya, T. Azuma, G. B.
(1990–1999). J. Antimicrob. Chemother. 46:1029–1031.
Nair, and D. E. Berg. 2000. Distinctiveness of genotypes of Helicobacter
9. Chopra, I., and M. Roberts. 2001. Tetracycline antibiotics: mode of action,
pylori in Calcutta, India. J. Bacteriol. 182:3219–3227.
applications, molecular biology, and epidemiology of bacterial resistance.
30. Nikaido, H. 2001. Preventing drug access to targets: cell surface permeability
Microbiol. Mol. Biol. Rev. 65:232–260.
barriers and active efflux in bacteria. Semin. Cell. Dev. Biol. 12:215–223.
10. Cover, T. L., D. E. Berg, M. J. Blaser, and H. L. T. Mobley. 2001. H. pylori
31. Osato, M. S., R. Reddy, S. G. Reddy, R. L. Penland, H. M. Malaty, and D. Y.
pathogenesis, p. 509–558. In E. A. Groisman (ed.), Principles of bacterial Graham. 2001. Pattern of primary resistance of Helicobacter pylori to met-
pathogensis. Academic Press, Inc., New York, N.Y.
ronidazole or clarithromycin in the United States. Arch. Intern. Med. 161:
11. Cuchi-Burgos, E., M. Forne Bardera, S. Quintana Riera, J. Lite Lite, and J.
Garau Alemany. 2002. Sensitivity of 235 strains of Helicobacter pylori from
32. Pioletti, M., F. Schlunzen, J. Harms, R. Zarivach, M. Gluhmann, H. Avila,
1995 to 1998 and impact of antibiotic treatment. Enferm. Infecc. Microbiol.
A. Bashan, H. Bartels, T. Auerbach, C. Jacobi, T. Hartsch, A. Yonath, and F.
Clin. 20:157–160.
Franceschi. 2001. Crystal structures of complexes of the small ribosomal
12. Debets-Ossenkopp, Y. J., A. J. Herscheid, R. G. Pot, E. J. Kuipers, J. G.
subunit with tetracycline, edeine and IF3. EMBO J. 20:1829–1839.
Kusters, and C. M. Vandenbroucke-Grauls. 1999. Prevalence of Helicobacter
33. Quinones, M., J. E. Knesek, and S. A. McIntire. 2001. Sequence and gene
pylori resistance to metronidazole, clarithromycin, amoxycillin, tetracycline expression analyses of plasmid pHPM8 from Helicobacter pylori reveal the and trovafloxacin in The Netherlands. J. Antimicrob. Chemother. 43:511–
presence of two operons with putative roles in plasmid replication and antibiotic activity. Plasmid 46:223–228.
13. Dubois, A., D. E. Berg, E. T. Incecik, N. Fiala, L. M. Heman-Ackah, J. Del
34. Ross, J. I., E. A. Eady, J. H. Cove, and W. J. Cunliffe. 1998. 16S rRNA
mutation associated with tetracycline resistance in a gram-positive bacte- P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete
rium. Antimicrob. Agents Chemother. 42:1702–1705.
genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:
35. Ross, J. I., A. M. Snelling, E. A. Eady, J. H. Cove, W. J. Cunliffe, J. J. Leyden,
P. Collignon, B. Dreno, A. Reynaud, J. Fluhr, and S. Oshima. 2001. Pheno-
42. Trieber, C. A., and D. E. Taylor. 2002. Mutations in the 16S rRNA genes of
typic and genotypic characterization of antibiotic-resistant Propionibacterium Helicobacter pylori mediate resistance to tetracycline. J. Bacteriol. 184:2131–
acnes isolated from acne patients attending dermatology clinics in Europe, the USA, Japan and Australia. Br. J. Dermatol. 144:339–346.
43. Versalovic, J., M. S. Osato, K. Spakovsky, M. P. Dore, R. Reddy, G. G. Stone,
36. Sisson, G., J. Y. Jeong, A. Goodwin, L. Bryden, N. Rossler, S. Lim-Morrison,
D. Shortridge, R. K. Flamm, S. K. Tanaka, and D. Y. Graham. 1997. Point
A. Raudonikiene, D. E. Berg, and P. S. Hoffman. 2000. Metronidazole acti-
mutations in the 23S rRNA gene of Helicobacter pylori associated with different vation is mutagenic and causes DNA fragmentation in Helicobacter pylori and levels of clarithromycin resistance. J. Antimicrob. Chemother. 40:283–286.
in Escherichia coli containing a cloned H. pylori rdxAϩ (nitroreductase) gene.
44. Versalovic, J., D. Shortridge, K. Kibler, M. V. Griffy, J. Beyer, R. K. Flamm,
J. Bacteriol. 182:5091–5096.
S. K. Tanaka, D. Y. Graham, and M. F. Go. 1996. Mutations in 23S rRNA
37. Sisson, G., A. Goodwin, A. Raudonikiene, N. J. Hughes, A. K. Mukho-
are associated with clarithromycin resistance in Helicobacter pylori. Antimi- padhyay, D. E. Berg, and P. S. Hoffman. 2002. Enzymes associated with
crob. Agents Chemother. 40:477–480.
reductive activation and action of nitazoxanide, nitrofurans, and metronida- 45. von Recklinghausen, G., C. Di Maio, and R. Ansorg. 1993. Activity of anti-
zole in Helicobacter pylori. Antimicrob. Agents Chemother. 46:2116–2123.
biotics and azole antimycotics against Helicobacter pylori. Zentbl. Bakteriol.
38. Street, M. E., P. Caruana, C. Caffarelli, W. Magliani, M. Manfredi, F.
Parasitenkd. Infektkrankh. Hyg. Abt. 1 Orig. 280:279–285.
Fornaroli, and G. L. de’Angelis. 2001. Antibiotic resistance and antibiotic
46. Wang, G., and D. E. Taylor. 1998. Site-specific mutations in the 23S rRNA
sensitivity based treatment in Helicobacter pylori infection: advantages and gene of Helicobacter pylori confer two types of resistance to macrolide- outcome. Arch. Dis. Child. 84:419–422.
lincosamide-streptogramin B antibiotics. Antimicrob. Agents Chemother.
39. Takeuchi, H., M. Shirai, J. K. Akada, M. Tsuda, and T. Nakazawa. 1998.
42:1952–1958.
Nucleotide sequence and characterization of cdrA, a cell division-related 47. Westblom, T. U., S. J. Czinn, and J. G. Nedrud. 1999. Gastroduodenal
gene of Helicobacter pylori. J. Bacteriol. 180:5263–5268.
disease and Helicobacter pylori: pathophysiology, diagnosis and treatment.
40. Tillenburg, B., S. Siehoff, T. Becker, U. Peitz, G. Borsch, and J. Labenz.
Curr. Top. Microbiol. Immunol. 241.
1997. Helicobacter pylori: pretherapeutic resistance status in Germany (Ruhr 48. Wu, H., X. D. Shi, H. T. Wang, and J. X. Liu. 2000. Resistance of Helicobacter
area). Z. Gastroenterol. 35:165–169.
pylori to metronidazole, tetracycline and amoxycillin. J. Antimicrob. Chemo- 41. Tomb, J. F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D.
ther. 46:121–123.
Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K.
49. Yamaoka, Y., M. S. Osato, A. R. Sepulveda, O. Gutierrez, N. Figura, J. G.
Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D.
Kim, T. Kodama, K. Kashima, and D. Y. Graham. 2000. Molecular epide-
Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M.
miology of Helicobacter pylori: separation of H. pylori from East Asian and Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne,
non-Asian countries. Epidemiol. Infect. 124:91–96.
T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman,
50. Zgurskaya, H. I., and H. Nikaido. 2000. Multidrug resistance mechanisms:
C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky,
drug efflux across two membranes. Mol. Microbiol. 37:219–225.

Source: http://ww.kaunoklinikos.lt/klinika10/publikacijos/Emergence%20of%20tetracycline%20resistance%20in%20Helicobacter%20pylori.%20Multiple%20mutation%20changes%20in%2016S%20ribosomal%20DNA%20and%20other%20genetic%20loci.pdf